Resolution of dl-tryptophane and dl-alpha-phenylethylamine



Jan. 26, 1965 R, ovERBY 3,167,555

RESOLUTION OF DL-TRYPTGPHANE AND -a-PHENYLETHYLAMINE Filed oct. 1s, 1961 Inventor Overb,

j-Hiarne/ United States Patent O 3,167,566 RESLUTIGN @F DL-TRYPTPHANE AND dll-a-PHENYLTTHYLAMENE Lacy R. tlverhy, Waukegan, lib, assigner to Abbott Laboratories, North Chicago, lil., a corporation of Illinois Filed Get. 18, 1962i, Ser. No. idlil S Claims. (Cl. 269-319) This invention relates to the resolution of N-acetyl-DL- tryptophane to obtain L-tryptophane and other active components. In particular, it relates to a method which employs N-acetyl-DL-tryptophane and resolving agents obtained by reciprocal resolution of dl-a-phenylethylamine with N-acetyl-L-tryptophane. i

L-tryptophane is an active essential amino acid which is desirably a component in amino acid preparations for parenteral administration. lt is well known that protein hydrolysates prepared for nutritional therapy comprise a mixture of amino acids. lt is also Well known that hydrolysis of protein preparations under acid conditions decomposes L-tryptophane; therefore, it is a common practice to add -to such a protein hydrolysate the optically inactive DL-tryptophane. The L-form of the DL acid is beneficially utilized by the body While the D-form is inert or may possibly be detrimental. It is preferred to add L-tryptophane rather than the IDL-form to such hydrolysates, but L-tryptophane is not easily available and is very expensive. Therefore, it is desirable to ind a method which will produce L-tryptophane easily, economically and in large yields.

Nutritional therapy is one of the more obvious uses of Ithe optically active amino acid, but there are additional reasons why large, economical supplies of the active forms are desirable. Among such reasons is the requirement that both L- and D-coniigurations of tryptophane are necessary for studies of animal nutrition, enzyme actions, microbial metabolism, peptide synthesis and others.

The resolution of N-acetyl-DL-tryptophane to its optically active forms, also referred to as antipodes of the racemic mixture, has been practiced in the art. Such practice suffers from many disadvantages such as low yields, tedious crystallizations, expensive resolving agents and other problems.

It has been shown by du Vigneaud et al., 1. Biol. Chem. 96, 511 that when N-acetyl-DL-tryptophane is combined with an equivalent amount `of d-phenylethylamine, the less soluble diastereoisomeric salt of N-acetyl- D-tryptophane and d-e-phenylethylamine'was obtained in low yields by fractional crystallizations. The dextro form of the acid Was then liberated from the diastereoisomeric salt with a strong base. ln accordance'vvith this scheme, one mole of an expensive and hard-to-obtain dextro base is required to produce one-half mole of the dextro acid.

It is, therefore, apparent that great advantages woul accrue from a method for resolving N-acetyl-DL-tryptophane which provides an inexpensive resolving agent to complement the resolving action of smaller amounts of the expensive d-e-phenylethylamine. It is also apparent that an improved method results Where means are provided to obtain large supplies of d-a-phenylethylamine by economical and simple steps. Because L-tryptophane is the more useful isomer it is highly important to provide economically large quantities of l-a-phenylethylamine.

It is, therefore, an object of this inventionto provide an economic land simple means for resolving N-acetyl- DL-tryptophane and dl--phenylethylaniine.

Another object of this invention is to produce L-tryprtophane from readily available, optically inactive N-ace'tyl- DL-tryptophane by a method that employs the undesirable D-form in process steps to obtain the desired L-torrn.

Still another object of this invention is to provide a i,l7,566 Patented Jan. 26 1965 method of preparing large amounts of L-tryptophane by reciprocal resolution of optically inactive acetyl-DL- tryptophane with small amounts of l-e-phenylethylamine.

A still further object of this invention is to provide a method for obtaining `large amounts of the resolving agent, l-a-phenylethylamine by reciprocal resolution of dl-e-phenylethylamine with small amounts of acetyl-L- tryptophane.

Accordingly, it has been found that by complementing the action of the antipodes with optically inactive acid or base important advantages are realized. Optically inactive complementary agents aretaken to mean acids or bases that are molecularly symmetrical and thereby nonresolvable, thus excluding racemic mixtures.

It has now been found that various salts are formed by reacting one equivalent of the optically inactive acid, N- acetyl-DL-tryptophane, hereinafter designated as DL-A (see flow sheet in drawing), With one-half equivalent of the levo-active base of a-phenylethylamine, hereinafter designated as lB, in a solvent containing one-halt equivalent or" an optically Vinactive alkaline agent, hereinafter designated as M+. The least-soluble salt, LAlB crystallizes out of solution and is subsequently decomposed with an alkaline agent containing a cati-on (M+) to obtain the salt IvlLA. This latter form is hydrolyzed and then neutralized to obtain the desired L-tryptophane. The active base 1B may be recovered and employed again in the reaction. The more soluble MDA remaining in the filtrate from the LAlB crystallization is then racemized to give DLA which is, thereafter, resolved to give additional LA. By this process all the DLA is eventually converted to LA.

A key to the successful production of LA is a plentiful supply of 1B. ln order to obtain this active base, the practice of the process includes a reciprocal resolution wherein the active acid, LA, is used to resolve the dl base to obtain the active lB compound which is subsequently used for further resolution. This reciprocal resolution is conducted by neutralizing a portionk of MLA to LA, which is then combined with the dl-a-phenylethylamine or dlB. In the practice of this step, smaller amounts of optically active LA are employed to complement the easily available, optically inactive acid agents. Thus, thereaction is carried out with one-half equivalent LA and one equivalent dlB in a solvent containing one-half` equivalent mineral acid (HX). The less-soluble LAlB crystallizes from solution leaving Xdb in solution. Treatment with an optically inactive base providing an alkali metal cation (M+) decomposes LAlB and large` amounts of MLA and 1B are recovered. The collected salt MLAKis hydrolyzed and neutralized to obtain the desired L-tryptophane. The active base LB is separated and employed for the reaction with N-acetyl-DL-tryptophane as described hereinbefore.

The process set out herein operates elliciently and in a surprising manner by utilizing optically inactive alkaline agents and acid agents to complement the resolving action of the antipodes. It will be obvious to the skilled practitioner that alkaline agents, by their inherent properties, will react with the acid tryptophane. Such optically inactive alkaline agents can be selected, for example, from alkali metal and alkaline earth metal hydroxides such as sodium and calcium hydroxide among others, from alcoholates such as sodium ethylate and others or from strong organic bases such as butylarnine, dimethylamine, cyclohexylamine, tribenzylamine hydroxide and the like. It is further apparent that acid agents, by their inherent properties, will react with the base, dl-a-phenylethylamine. Among the optically inactive acid agents that can be used are mineral acids such as hydrochloric, sulfuric, .nitric and the like and organic acids such as acetic and the like. In the actual practice of the invention it is preferred to utilize a strong alkali metal base as sodium hydroxide and a strong mineral acid as hydrochloric acid.

aievgeee According tothe foregoing process, a small amount of LA or 1B can be employed to conveniently produce large quantities ofv L-tryptophane from N-acetyl-DL- tryptophane by the respective application of the reciprocal resolution of acid and base.

The -attached schematic drawing sets out theprocess in operation. It will be understood more fully after considering the following examples which setk out the practice of the invention in greater detail.

The followingr examples are presented to teach the method in operation, but it should be understood that said examples` are not intended to be an exclusive illustration of the method.

Example I To theV warm solution is added 60.5 gm. f

yield of 134 gm. (73%). The product has a specific rota-v .tion of [alD=-l-l7.80. The iiltrate is set aside for later Vtreatment as described in Example II.

The collected salt is suspended in about 250 cc. of water and about 50 cc. of benzene. The mixture is made alkaline to phenolphthalein with sodium hydroxide. The aqueous phase is separated and washed with 50 cc. portions of benzene. The combined benzene extracts are washed'with water and said water Wash is combined with the aqueous phase. This aqueous phase contains the sodium salt ,of N-acetyl-L-tryptophane which is treated to obtain L-tryptophane as hereinafter described or is reserved for reciprocal resolution of dl-m-phenylethylamine as described in Example III. The benzene phase contains -l-oc-phenylethylamine which is separated by drying the benzenefextracts and distillingvthrough a short column. The aqueous phase containing N-acetyl-L-tryptophane is adjusted with three equivalents of hydrochloric acid until its acidity is 2N. The solution is refluxed for four hours, decolorized with carbon and evaporated to dryness under reduced pressure. The residue is extracted with 95% ethanol to separate L-tryptophane hydrochlo-L ride from the sodium chloride.v The alcoholic solution is neutralized Vwith ammonium hydroxide to precipitate L- tryptophane. ltration, washed with water and alcohol, `then dried. The yield .is 95% and the specific rotation is [alD25=-31.2.

Example'll' The ltrate reserved in Example I is evaporated to dryness, and the residue is dissolved in about 250 cc. of water. The residue comprisesV basic Vsalts rich in N-acetyl-D- tryptophaneand some diastereoisomeric saltof N-acetyl- L-tryptophane and l-a-phenylethylamine. The mixture is made basic to phenolphthalein with sodium hydroxide to decompose the small portion of the diastereoiso-meric salt. The basic mixture is extracted with about 250 cc. of benzene to remove the liberated amine. The aqueous phase is decolorized with activated carbon and 150 cc. of acetic anhydride is added. The solution is seeded with N-acetyl-DL-'tryptophane and kept at 40l C. overnight, whereupon N-acetyl-DL-tryptophane crystallizes from the solution in about a 92% yield. The mixture is chilled and the crystalline product is removed by filtration, washed with water and dried. Specific rotation [M1325 is Zero, M P. 20S-206 C. The racemic N-acetyl-DL-tryptophane is set aside for further resolution by l--phenylethylamine.

`Example III Thefprecipitated product-is removed by bon and acidied to pH 3 with hydrochloric acid. Pollowing this neutralization, N-acetyl-L-tryptophane precipitates in a yield of 96%. The solid product is sepa;

mated by filtration :and washed with water. The specific rotation is [11]]325: +291 The collected N-acetyl-LJtryptophane (123 gm.), (0.5 mole) is dissolved in 250 cc..of warm ethanol. To f this solution is added 0.5 mole of concentrated hydrochloric acid followed by 121 gm. (1.0 mole) of dl-ephenylethylamine. The solution is seeded with LAlB crystalsland allowedto crystallize at room temperature.

The crystalline product is the diastereoisomeric salt of N-acetyl-L-tryptophane and 'l-a-phenylethylamine which is collected ina yield of 151 gm. (83%), having a specific rotation of []D25=-1l7.7. is suspended in 25 cc. of water and 50 cc. benzene and the mixture is vmade alk'alinewith sodium hydroxide to phenolphthalein. Thesodium salt of N-acetyl-L-tryptophane` is obtained inthe aqueous phase and Lor-phenylethylainine is present in the water-immiscible phase (benzene). The l-m-phenylethylamine in the benzene phasev is recovered as an oil by followingthe procedural steps set'out in Example I.' The collected l-a-phenylethylamme has -a boiling point of 18S-187 C. and a specific rotation of [a]D25=-38.8 to 39.3. The l-u-phenylethylamine collected in this example .is reserved for fur-ther resolu-.

tionof N-acetyl-DL-tryptophane las described in Example I. The lsodium salt of N-acetyl-L-trypftophane is reserved for conversion to L-tryptophane as describedr in Example I, or re-used to resolve more dl-u-phenylethylamine.

The process set out inthe `foregoing examples is schematically represented in the attacheddrawing By refierence vtosuch drawing, the advantages of the process become apparent. Under the subdivision designated l, the initial step shows the combination of N-acetyl-DL- tryptophane, (DLA) l-fx-phenylethylamine (1B) and an alkaline agent (M+). This combination producesthe antipode salt MDA and therdiastereoisomeric salt LAlB. The antipode salt MDA `remains in solution with about 27% of the formed diastereoisomeric salt LAlB. The

major portion of the less-soluble LAlB precipitates from solution as indicated by the heavy arrow. Treatment of the collected LAlB with an alkaline agent gives the anti-v pode lB in an organic solvent phase and the antipode salt MLA in an Vaqueous phase. The antipode salt MLA is hydrolyzed and neutralized to provide the antipode, L-

tryptophane or LA. The rantipode lBis removed from the organic solvent `phase and is reserved for the initial and reserved lfor the resolution of DLA. The antipode salts MDA and MLA are racemized in the aqueous phase to form DLA which is .reservedY for the initial step as shown by the broken arrow.

Subdivision ll of the drawing shows the reciprocall resolutionof dl--phenylethylamine with one-half equivalents of the antipode LA andan optically inactive acid agent HX. The antipode LA is obtained by treating with an acid agent la portion or all of the antipode salt MLA prepared under Subdivision l. The obtained ,antipode LA is then comb-ined with the racemicdlB and acid agentk HX to obtain the antipode salt XdB and the diastereoisomeric salt LAlB. The major portion of the less-soluble LAlB precipitates from solution as shown by the: heavy arrow. The precipitated LAlB is then treated with anoptically inactive yalkaline agent 'M+ to formy additional amounts of the ,antipode 1B `and the antipode sait MLA.

The solution containing fasmalier portion (17%) of the- Vdiastereoisomerie salt LAlB and the antipode salt XdB is treated with an optically inactive alkaline agent M+ The diastereoisomeric salt to form the lantipode salt MLA and the antipodes dB and 1B.

By employing the reciprocal resolution of this method according to the examples and as shown in the schematic drawings, large quantities of L-tryptophane are obtained readily. Thus, `by starting out with 60.5 gm. of the antipode l-a-phenylethylamine and a plentiful supply of the racemic compounds, NJacetyl-DL-tryptophane and d1- a-phenylethylamine, 12.5 kg. of acetyl-L-tryptophane and 8.6 kg. `of l-fx-phenylethylamine is realized after 17 reciprocal resolutions.

It will be appa-rent to the skilled practitioner in the art that the `advantages of this method are easily adapted to a continuous process, in which small amounts of starting active material and large amounts of easily available inactive material are continuously manipulated by reciprocal resolution to obtain large amounts of the desired L-tryptophane. It is equally apparent that D-tryptophane, d-a-phenylethylamine and their derivatives can be obtained with the foregoing process steps when acetyl-D- tryptophane and d-a-phenylethylamine are used as the active agents to resolve the optically inactive acid and base.

The source of racemic tryptophane employed herein is N-acetyl-DL-tryptophane, but it will be apparent that other N-acyl derivatives of racemic tryptophane are equally operable in the process such as N-propionyl, N- butyryl, N-amylyl and the like. The foregoing acyl groups are easily removed by hydrolysis `and neutralization following resolution of the racemic Iform.

This application is a continuation-impart of co-pending application Serial No. 757,253 led August 26, 1958, now abandoned.

Others may practice the invention in any of the numerous ways which will be suggested by this disclosure to one skilled in the tart. All such practice of the invention is considered to be a part hereof provided it falls within the scope of the appended claims.

I claim:

1. In the resolution process wherein an optically active form of amine resolving agent and N-lower-alkanoyl-DL- tryptophane are reacted to obtain a diastereoisomeric salt of said tryptophane and amine agent which is then decomposed with a strong base to liberate the dextro tryptophane form from the diastereoisomeric salt the improvement comprising complementing the amine resolving agent by combining one equivalent of an optically inactive base with about two equivalents of N-lower alkanoyl-DL-tryptophane and about one equivalent of L-a-phenylethylamine to obtain diastereoisomeric salt which is capable of decomposition to yield the dextro tryptophane form.

2. In the process of resolving N-acetyl-DL-tryptophane wherein L--phenylethylamine is employed as a resolving agent the improvement comprising combining two moles of N-acetyl-DL-tryptophane with about one mole of optically inactive base capable of forming a soluble salt, and about one mole of said L-oa-phenylethylamine to obtain a soluble product and an insoluble product, said soluble product essentially consisting of diastereoisomeric salts of N-acetyl-L-tryptophane and of N-acetyl-dextro tryptophane, said insoluble product essentially consisting of the diastereoisomeric salt of N-acetyl-L-tryptophane and L-aphenylethylamine.

3. In the process of resolving DL--phenylethylamine wherein N-acetyl-L-tryptophane is used as a resolving agent the improvement comprising combining two equivalents of said amine with about one equivalent of said L- tryptophane and about one equivalent of optically inactive acid to form diastereoisomeric salt of N-acetyl-L- tryptophane and L--phenylethylamine, adding alkali to the formed salt to cause decomposition thereof to yield L-a-phenylethylamine and alkali salt of N-acetyl-L-tryptophane.

4. The process of claim 1 wherein said optically inactive base is alkaline metal hydroxide.

5. The process of claim 3 wherein said acid is mineral acid.

Du Vigneaud et al.: I. Biological Chem., vol. 96, pp. 511 (1932).

AHackhs Chemical Dictionary, 2nd Ed., Maple Press Co., 1937, page 21. 

1. IN THE RESOLUTION PROCESS WHEREIN AN OPTICALLY ACTIVE FORM OF AMINE RESOLVING AGENT AND N-LOWER-ALKANOYL-DLTRYPTOPHANE ARE REACTED TO OBTAIN A DIASTEREOISOMERIC SALT OF SAID TRYPTOPHANE AND AMINE AGENT WHICH IS THEN DECOMPOSED WITH A STRONG BASE TO LIBERATE THE DEXTRO TRYPTOPHANE FORM FROM THE DIASTEREOISOMERIC SALT THE IMPROVEMENT COMPRISING COMPLEMENTING THE AMINE RESOLVING AGENT BY COMBINING ONE EQUIVALENT OF AN OPTICALLY INACTIVE BASE WITH ABOUT TWO EQUIVALENTS OF N-LOWER ALKANOYLDL-TRYPTOPHANE AND ABOUT ONE EQUIVALENT OF L-A-PHENYLETHYLAMINE TO OBTAIN DIASTEREOISOMERIC SALT WHICH IS CAPABLE OF DECOMPOSITION TO YIELD THE DEXTRO TRYPTOPHANE FORM.
 3. IN THE PROCESS OF RESOLVING DL-A-PHENYLETHYLAMINE WHEREIN N-ACETYL-L-TRYPTPHANE IS USED AS A RESOLVING AGENT THE IMPROVEMENT COMPRISING COMBINING TWO EQUIVALENTS OF SAID AMINE WITH ABOUT ONE EQUIVALENT OF SAID LTRYPTOPHANE AND ABOUT ONE EQUIVALENT OF OPTICALLY INACTIVE ACID TO FORM DIASTEREOISOMERIC SALT OF N-ACETYL-LTRYPTOPHANE AND L-A-PHENYLETHYLAMINE, ADDING ALKALI TO THE FORMED SALT TO CAUSE DECOMPOSITION THEREOF TO YIELD L-A-PHENYLETHYLAMINE AND ALKALI SALT OF N-ACETYL-L-TRYPTOPHANE. 